These extracts were tested against two mammalian viruses, herpes simplex virus HSV-1 and vesi-cular stomatitis virus VSV, using Vero cells as the cell culture system, and two marine viru
Trang 1R E S E A R C H Open Access
In vitro evaluation of marine-microorganism
extracts for anti-viral activity
Jarred Yasuhara-Bell1,2, Yongbo Yang2, Russell Barlow3, Hank Trapido-Rosenthal3, Yuanan Lu1,2*
Abstract
Viral-induced infectious diseases represent a major health threat and their control remains an unachieved goal, due
in part to the limited availability of effective anti-viral drugs and measures The use of natural products in drug manufacturing is an ancient and well-established practice Marine organisms are known producers of pharmacolo-gical and anti-viral agents In this study, a total of 20 extracts from marine microorganisms were evaluated for their antiviral activity These extracts were tested against two mammalian viruses, herpes simplex virus (HSV-1) and vesi-cular stomatitis virus (VSV), using Vero cells as the cell culture system, and two marine virus counterparts, channel catfish virus (CCV) and snakehead rhabdovirus (SHRV), in their respective cell cultures (CCO and EPC) Evaluation of these extracts demonstrated that some possess antiviral potential In sum, extracts 162M(4), 258M(1), 298M(4), 313 (2), 331M(2), 367M(1) and 397(1) appear to be effective broad-spectrum antivirals with potential uses as prophylac-tic agents to prevent infection, as evident by their highly inhibitive effects against both virus types Extract 313(2) shows the most potential in that it showed significantly high inhibition across all tested viruses The samples tested
in this study were crude extracts; therefore the development of antiviral application of the few potential extracts is dependent on future studies focused on the isolation of the active elements contained in these extracts
Background
Viruses cause many important diseases in humans, with
viral-induced emerging and re-emerging infectious
dis-eases representing a major health threat to the public In
addition, viruses can also infect livestock and marine
spe-cies, causing huge losses of many vertebrate food species
Effective control of viral infection and disease has
remained an unachieved goal, due to virus’ intracellular
replicative nature and readily mutating genome, as well as
the limited availability of anti-viral drugs and measures
The use of natural products in the manufacturing of
drugs is an ancient and well-established practice that
has yielded such familiar products as morphine, digitalis,
penicillin, and aspirin [1] Natural products derived
from terrestrial and marine kingdoms represent an
inex-haustible source of compounds with promising antiviral
action, not only for the great number of species found
in these kingdoms with unexplored pharmacological
activities, but mainly for the variety of synthesized
metabolites In relation to infectious diseases, the exploration of the marine environment represents a pro-mising strategy in the search for active compounds, whereas there is a need for new medicines, due to the appearance of resistance to available treatments in many microorganisms, specifically concerning antifungal, anti-protozoal, antibacterial and antiviral activities
The marine environment represents approximately half
of the global biodiversity and could provide unlimited bio-logical resources for the production of therapeutic drugs [1-3] Almost all forms of life in the marine environment (e.g algae, sponges, corals, ascidians) have been investi-gated for their natural product content [4] Ecological pressures, such as competition for space, predation, sym-biosis and tide variations, throughout thousands of years, originated the biosynthesis of complex secondary metabo-lites by these organisms, which in turn, allowed their adap-tation to a competitive and hostile environment [3] The first serious work on marine organisms started only 50 years ago In the following 50 years, marine organisms (algae, invertebrates and microbes) have pro-vided key structures and compounds that proved their potential for industrial development as cosmetics, nutri-tional supplements, fine chemicals, agrochemicals and
* Correspondence: ylu@pbrc.hawaii.edu
1 Department of Tropical Medicine, Medical Microbiology and Pharmacology,
John A Burns School of Medicine, University of Hawaii at Manoa, 651 Ilalo
Street, BSB Suite 320, Honolulu, HI, 96813, USA
Full list of author information is available at the end of the article
© 2010 Yasuhara-Bell et al; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
Trang 2therapeutic agents for a variety of diseases Some
exam-ples of commercially available marine bioproducts that
have been developed include: a) Ara-A (vidarabine) and
Ara-C (cytarabine) (antiviral drugs) derived from the
sponge Tethya cripta; b) Okadaic acid and Manoalide
(molecular probes) from Dinoflagellate and the sponge
Luffariella variabilis, respectively; c) Green Fluorescent
Protein (GFP, Reporter gene) from the jellyfish Aequora
victoria; d) Phycoerythrin (conjugated antibodies) used
in Enzyme-Linked ImmunoSorbent Assays (ELISA) and
flow cytometry from red algae and; e) Pseudopterosins
(additives in skin crèmes) from the soft coral
Pseudop-terogorgia elizabethae [1] As a result, important
phar-macological and therapeutic products are currently
being obtained and actively sought from the ocean
[1,2,4-21]
The current antiviral drug armamentarium comprises
over 40 compounds that have been officially approved
for clinical use, with at least half of them being used to
treat HIV infection [1,3,17] Marine antiviral agents
(MAVAs) [22] can be used for the biological control of
human enteropathogenic virus contamination and
dis-ease transmission in sewage-polluted waters, as
che-motherapy for viral diseases of humans and lower
animals, as well as the biological control of viral diseases
of marine animals The seeding of MAVAs under
nat-ural conditions, or when marine mammals are kept in
captivity for various uses, could control viral disease
transmission within these select populations It is clear
that the marine environment will play a vital role in the
future development and trials of anti-infective drugs
Within the Environmental Health Laboratory at the
University of Hawai’i at Manoa, four representative
viruses isolated from mammal-and marine-animal
spe-cies were collected and prepared In addition, a cell line
bank was established, comprising over 150 cell lines
derived from various organs and tissues of different
ani-mal species Also, over 2,000 unpurified crude extracts
from a variety of marine organisms, including sponges,
bacteria and algae, have been prepared in Dr Thomas
Hemscheidt’s laboratory at the University of Hawai’i at
Manoa These compounds and extracts were initially
being tested for anti-bacterial and anti-tumor activities
The purpose of this study was to establish an in vitro
model to screen marine extracts for antiviral activity
and to evaluate 20 marine extracts for their antiviral
potential, with a long-term goal of discovering new
mar-ine compounds to be used as potential antiviral drug
candidates
Methods
Cell Cultures
Readily available cell cultures essential for supporting
viral infectivity of the test viruses (Table 1) were used in
this study Green African monkey kidney (Vero) cells (ATCC®, Manassas, VA, Cat No CCL-81™) and Epithe-lioma papulosum cyprini (EPC), carp skin cells (ATCC®, Manassas, VA, Cat No CRL-2872™) were grown with Eagle’s minimal essential medium (MEM) (Sigma-Aldrich, St Louis, MO) supplemented with 10% heat-inactivated bovine calf serum (BCS) (HyClone, Logan, UT) and 1% GPS solution (100 U/mL penicillin, 100 μg/
mL streptomycin sulfate, and 4 mM L-glutamine: Sigma-Aldrich, St Louis, MO) at 37°C with humidified 5.0% CO2 and at room temperature (23 ± 1°C) under normal atmospheric conditions, respectively Chanel cat-fish ovary (CCO) cells (ATCC®, Manassas, VA, Cat No CRL-2772™) were grown with high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St Louis, MO) supplemented with 10% heat-inactivated standard fetal bovine serum (FBS) (HyClone, Logan, UT) and 1% GPS solution at room temperature
Cells were subcultured at a 1:3 ratio every 3-4 days Briefly, media from TC-75 cm2 flasks were collected and centrifuged at 3000 rpm for 5 minutes Meanwhile, 5.5 ml/flask of a trypsin-versine solution (10 ml 10 × Tryp-sin (Sigma-Aldrich, St Louis, MO) in 90 ml pre-steri-lized versine (EDTA) solution) was added to detach the cell monolayer [23] Following cell detachment, cleaned medium was added back into the flasks to neutralize the trypsin activity The contents of the flasks were then removed and centrifuged at 1000 rpm for 5 minute Fol-lowing centrifugation, supernatant was removed and new growth medium was used to resuspend the cells Cells, split 1:3, were placed back into flasks and total media volume was brought up to 10 ml/flask Flasks were then placed back into their respective incubators and monitored daily The pH of the medium was moni-tored and adjusted to 7-7.5 using HEPES buffer (Media-tech, Herndon, VA) or 7.5% w/v NaHCO3 (Mediatech, Herndon, VA)
Viruses
The viral isolates used in this study (Table 1) are avail-able in the laboratory and methodologies for their repli-cation and purifirepli-cation, as well as quantitative infection assays, have been established and routinely used [24] These representative indicator viruses were propagated and quantified as viral stocks for this study Briefly, cells were grown and seeded into TC-75 cm2 flasks, as pre-viously described, so that an approximately 90% cell monolayer formed in 24 hours All medium was removed from the flask and 250 μl of previously made virus stock was mixed with 2 ml of serum-free medium and added into the flask to infect the cells The flasks were incubated for 1 hour and then inoculum was removed Cells were washed twice with serum-free med-ium and then 10 ml of medmed-ium supplemented with 5%
Trang 3serum was added into the flask The flasks were then
incubated at the optimal temperature for viral
replica-tion, until the visual appearance of approximately 90%
cytopathic effects (CPE) (rounding of cells, loss of
con-tact inhibition and cell death), after which the flasks
were stored at -80°C for 24 hours Following two cycles
of the freeze-thaw, the contents of the flasks were
com-pletely harvested and centrifuged at 1000 rpm for 5
minutes to remove all cellular debris Supernatant was
then collected and aliquots of 0.5 ml/tube were stored
long-term at -80°C or short-term at -20°C Viral titers
were determined using plaque assays, as described
below
Extracts
Twenty marine-microorganism extracts were tested for
their antiviral activities in this study These extracts
were provided from Dr Thomas Hemscheidt’s
labora-tory at the University of Hawai’i at Manoa (Table 2)
Microbial colonies were collected from sites around the
Hawaiian Islands and various sites in the open ocean
Briefly, cultures were isolated, made axenic, identified by
16 s ribosomal DNA (rDNA) PCR, classified, and
sub-mitted for culturing Upon receipt, each culture was
given a Center for Marine Microbial Ecology and
Diver-sity (CMMED) number and cryogenically frozen in
quartet (if possible) An example of a CMMED# is as
follows: 288 (1), where the (1) denotes that this was the
first grow out of this particular culture and subsequent
grow outs of the same culture are denoted as (2), (3)
etc To harvest and extract marine bacteria, cultures
were spun down and pelleted at 5,000 g for 18-20 min
The supernatant was then extracted with ethyl acetate
and the pellet was extracted with 2:1 methylene
chlor-ide: 2-propanol Cultures that had both the media/
supernatant and pellet extracted are differentiated from
one another by the addition of an M to the CMMED#
to denote a media extraction (e.g CMMED# 288 M (1))
To extract diatoms, cyanobacteria, etc., entire cultures
skipped the harvesting and both the cells and media
were extracted with ethyl acetate Cultures that were
extracted without pelleting were given an M on the
extract number Solvent was then removed via overnight
speed vacuum The samples were then dissolved in
DMSO at a concentration of 100 mg/ml and then used for screening
Plaque Assay
Briefly, cells were cultured and then seeded into multi-well plates at density that would allow the formation of
an approximately 90% monolayer in 24 hours Once a confluent cell monolayer was formed, media from the wells was aspirated Meanwhile, serial 10-fold dilutions
of stock virus were made and 100μl/well of each viral dilution were added to the plates Plates were incubated for 1 hour, then inoculum from each well was comple-tely removed and 2 ml/well of a 0.75% (w/v) methylcel-lulose overlay medium, containing 5% serum and 1% GPS solution, was added Plates were then incubated for 3-4 days to allow viral plaque development Viral pla-ques were visualized by the addition of 2 ml/well of crystal violet staining solution for at least 2 hours [25] and vigorous washing with tap water Plaques were counted visually and the viral titer calculated as follows: Virus Titer (PFU/ml) = [# plaques counted × dilution factor’/amount of viral inoculum used (0.1 ml)
Cytotoxicity Assay
Briefly, cells were maintained, as previously described, and then seeded into 96-well plates at a density that would allow the formation of a 90% monolayer in 24 hours Once a confluent cell monolayer was observed, media from the wells was removed Each extract was diluted in medium supplemented with 5% serum, with subsequent DMSO dilutions used as controls For pur-poses of this study, four concentrations, including 100,
50, 25 and 12.5μg/ml, were tested Control dilutions of DMSO at 0.1%, 0.05%, 0.025% and 0.0125% were also included Then, 200 μl/well of diluted extract and DMSO controls were added to the plates, at 4 wells/ concentration, and then the plates were incubated for
3 days
A Methylthiazol Tetrazolium (MTT) assay commonly used for cell proliferation was adopted to test for cell viability In brief, following the 3-day incubation, 20 μl/ well of MTT (VWR, West Chester, PA) was added to each plate The plates were then incubated in a dark incubator for 2-4 hrs, with checking every 30 minutes
Table 1 Cell culture systems and representative viruses
Name Species of Origin Susceptable viruses Viral Family Host
Vero African Green
Monkey kidney
epithelial cells
HSV-1 (herpes simplex virus type 1) VSV (vesicular stomatitis virus)
Herpesviridae Rhabdoviridae
Mammalian EPC Cyprinis carp skin SHRV (snakehead rhabdovirus) Herpesviridae Marine
CCO Channel catfish ovary CCV (channel catfish virus) Rhabdoviridae
Trang 4for purple formazan crystal formation Once proper
for-mazan crystal formation was observed, the contents
from the wells were completely aspirated Immediately
after, 100 μl/well of 100% DMSO was added to each
plate and then incubated at room temperature on a
mixer for 30 minutes Absorbance at 570 nm was read
on a microplate reader (Beckman Coulter AD 340C,
Beckman Coulter, Fullerton, CA) Any extract producing
a 10% or more reduction in cell viability was considered
toxic
Viral Attachment/Entry Inhibition Assay
Cells at exponential growth phase were harvested and
seeded into multi-well plates at densities that would
allow the formation of an approximately 90% cell
mono-layer overnight Marine extracts were diluted with
serum-free medium to twice the effective safe
concen-trations, as determined by the cytotoxicity tests Viruses
were diluted in serum-free medium to optimum
concen-trations that would yield approximately 50-100 PFU/
well, as determined by previous plaque assays Then,
250 μl of each extract at twice the maximum nontoxic
concentration (e.g., 200 μg/ml for those found to be
nontoxic at 100μg/ml) was mixed with an equal volume
of the virus dilution Positive controls were made by
mixing 250μl of virus dilution with 250 μl of
serum-free medium with 0.2% DMSO, in order to yield a final DMSO concentration of 0.1% These 500 μl virus/ extract mixtures were pre-incubated for 1 hour, along with controls, and then assayed for viral infectivity using the optimized plaques assay protocols Extracts produ-cing a reduction in plaque formation were considered for further characterization Antiviral effect of each extract was categorized as having no meaningful inhibi-tion (< 20%), slight inhibiinhibi-tion (≥ 20%), moderate inhibi-tion (≥ 50%), or high inhibition (≥ 80%)
Viral Replication Inhibition Assay
Test cells were seeded into TC-12.5 cm2flasks (BD Fal-con, San Jose, CA) at a density that would allow the for-mation of an approximately 90% monolayer the next day Marine extracts were diluted with medium contain-ing 5% serum to their safe and effective concentrations,
as determined by the cytotoxicity tests Medium was completely aspirated from the flasks, and then the cell monolayer was briefly washed with DPBS, before infec-tion with test virus at a multiplicity of infecinfec-tion (MOI)
of 0.1 Following a 1-hr viral adsorption, all medium in the flask was removed and the flasks were washed twice with DPBS (Sigma-Aldrich, St Louis, MO) Infected cul-tures were incubated with 2.5 ml/flask of diluted extract Two flasks were tested per extract and these cultures
Table 2 Marine extracts and their antiviral effects
Extract Source Herpesvirus Rhabdovirus
Mammalian Marine Mammalian Marine HSV-1 CCV VSV SHRV 162M(4) Marine bacterium; unclassified +++ + +++ N/T 185M(4) Roseobacter sp + N/T ++ N/T 219M(3) Pseudoalteromonas sp + N/T +++ N/T 258M(1) Cyanobacterium; Blue-green algae +++ N/T +++ N/T 298M(2) Marine bacterium; unclassified +++ +++ +++ + 312(2) Marine diatom; cf Odontella sp.; Bacillariophyceae +++ N/T +++ N/T 313(2) Marine diatom; Amphora sp.; Bacillariophyceae ++ +++ +++ +++ 328(2) Marine diatom; cf Odontella sp.; Bacillariophyceae + N/T +++ N/T 331M(3) Shewanella frigidmarina + +++ - + 338(1) Bacillus methanolicus - N/T + N/T 338M(1) Bacillus methanolicus + N/T + N/T 367M(1) Marine bacterium; unclassified +++ N/T +++ N/T 388(1) Marine bacterium; unclassified - ++ + -397(1) Marine bacterium; unclassified - ++ +++ -397M(1) Marine bacterium; unclassified N/T +++ N/T + 438M(1) Marine bacterium; unclassified ++ N/T - -460(1) Marine bacterium; mixed - N/T ++ N/T 475(1) Marine bacterium; unclassified ++ N/T ++ N/T 476(1) Marine bacterium; Proteobacteria/Halomonas ++ N/T +++ N/T 491(1) Marine bacterium; unclassified - N/T - N/T 495M(1) Marine bacterium; unclassified ++ +++ ++ N/T
- = No meaningful inhibition (< 20%); + = Slight inhibition ( ≥ 20%); ++ = Moderate inhibition (≥ 50%); +++ = High inhibition (≥ 80%); N/T = not tested.
Trang 5were allowed to incubate for 3 days Pictures were taken
every 12 hrs using an inverted microscope equipped
with a camera (Nikon Eclipse TE2000-U), starting at
time zero, in order to track the progression of
viral-induced CPE To track viral progression, 200-μl samples
of medium were taken from each flask, every 12 hours,
and stored at -20°C until the end of the experiment
The viral titers of these samples were later determined
by standard plaque assay, as previously described Test
extracts shown to produce a visually noticeable
reduc-tion in CPE, as well as a reducreduc-tion in viral titer, were
considered for further characterization
Data Analyses
Using OriginPro 8 (OriginLab Corporation,
Northamp-ton, MA), a one-way ANOVA was performed on the
data to determine significance The alpha value was set
at 0.05 to yield a significance with > 95% confidence
Results
Extract Cytotoxicity
To properly assess these marine extracts for antiviral
activity, a set of experimental tests were performed to
determine the safe and effective dose of these extracts
to be used for each cell culture system Experimental
results revealed that extracts 298M(2), 313(2), 331M(3)
and 438M(1) were toxic to Vero cells at a dose of
100 μg/ml, with 298M(2) definitively being the most
toxic (P < 0.001), followed by 313(2), 331M(3) and
438M(1) (P < 0.05, P < 0.05 and P < 0.5, respectively)
(Table 3) These four extracts also showed varied levels
of cytotoxicity at a concentration of 50μg/ml, although
this apparent toxicity was far less, if not negligible, as
compared to that observed at a concentration of
100 μg/ml These observations are consistent with that
observed visually through a microscope To be safe,
these three extracts were used at a concentration of
25μg/ml in the latter experiments involving Vero cells
All other extracts were found to be nontoxic to Vero
cells at all tested concentrations and were therefore used at 100 μg/ml in the latter experiments involving Vero cells
Extract samples available in sufficient amounts were also tested for their cytotoxicities to CCO and EPC cells (Table 3) Again, the results of these cytotoxicity assays showed that nearly all the tested extracts were nontoxic
to CCO and EPC cells at the maximum tested concen-tration of 100 μg/ml Extracts 298M(2), 313(2) and 331M(3) were toxic to CCO cells at a dose of 100 μg/
ml, with 298M(2) definitively being the most toxic (P < 0.001), followed by 313(2) and 331M(3), which showed
an approximately equal toxicity (P < 0.01 and P < 0.005, respectively) These data are consistent with visual observations of cell morphology and presence using a microscope Therefore, these three extracts were used at
a concentration of 25 μg/ml in the latter experiments involving CCO cells Extract 298M(2) was the only extract found to be cytotoxic to EPC cells It was extre-mely cytotoxic, as gross cell death was easily visible with
a microscope, even at a concentration of 25μg/ml For this reason, this extract was used at a concentration of 12.5μg/ml in the latter experiments involving EPC cells
Viral Attachment/Entry Inhibition
Since little is known about the antiviral nature of these marine extracts at the beginning of these experiments, these extracts were first tested for their ability to block viral attachment/entry into the cells These twenty extracts exhibited different levels of inhibitory effect on viral plaque formation (Table 2, Figure 1) Approxi-mately 14 extracts showed different levels of antiviral impact against HSV-1 in Vero cells (Table 2): three [162M(4), 258M(1) and 367M(1)’ possessed high anti-viral activity (> 90%), seven [298M(2), 312(2), 313(2), 438M(1), 475(1), 476(1) and 495M(1)’ produced moder-ate inhibitory effects (≥ 50%) and another four [185M (4), 328(2), 331M(3) and 338M(1)’ produced slight inhi-bitory effects (≥ 20%), while the other 6 showed no effect
The tested extracts also showed varying levels of anti-viral impact against VSV in Vero cells (Table 2): five extracts [219M(3), 312(2), 313(2), 328(2) and 367M(1)’ showed high antiviral activity (> 80%), while eight other extracts [162M(2), 185M(4), 258M(1), 298M(2), 397(1), 460(1), 475(1) and 476(1)’ showed a moderate antiviral effect (≥ 50%) Extract 495M(1) showed slight inhibition, with inhibition being observed as viral plaque reductions
of 43%, while the other 6 showed no antiviral effect (< 20%)
Remaining available extracts were tested in CCO cells
to determine if they possessed any inhibitory effects towards marine herpes virus CCV (Table 2) Experimen-tal results show that four extracts [298M(2), 313(2),
Table 3 Summary of extract cytotoxicity
Cells Extract Extract Concentration
12.5 mg/ml 25 mg/ml 50 mg/ml 100 mg/ml
Vero 298M(2) - - + +
313(2) - - + +
331M(3) - - + +
438M(1) - - + +
CCO 298M(2) - - + +
313(2) - - + +
331M(3) - - + +
EPC 298M(2) - + + +
*Summary table of extracts showing toxicity All other extracts were nontoxic
at all tested extract concentrations.
Trang 6331M(2) and 397M(1)’ had high inhibitory effects
against CCV in CCO cells (> 90%) Extract 495M(1)
showed moderately high antiviral potential against CCV,
with ~90% inhibition, while extracts 388(1) and 397(1)
showed moderate antiviral activity, with ~70%
inhibi-tion Extract 162M(4) showed slight antiviral activity
(approximately 40% inhibition) The other tested
extracts showed no apparent antiviral activities (< 20%)
Remaining available extracts were also tested in EPC
cells to determine if they possessed any inhibitory effects
towards marine rhabdovirus SHRV (Table 2)
Experimen-tal results show that extract 313(2) was the only extract
producing high antiviral activity against SHRV in EPC
cells, with an inhibition of > 90% Three other extracts
[397M(1), 298M(2) and 331M(2)’ showed moderate to low
inhibitory properties towards SHRV in EPC cells, with
inhibition being ~50%, ~30%, and ~25%, respectively All
other tested extracts showed no apparent inhibition
Viral Replication Inhibition
In addition to viral attachment/entry, marine extracts
potentially possess other means of virus inhibition, such
as affecting viral replication after the cell is infected Therefore, an additional set of experiments were per-formed to determine if these extracts can inhibit virus replication Results from the viral replication inhibition experiments showed different patterns of antiviral activ-ity, under the described conditions (Figure 2) Extract 298M(2) was the only extract showing antiviral potential against HSV-1 Extract 298M(2) mediated HSV-1 repli-cation within 24 hours post-infection and this antiviral effect was evident throughout the duration of the experiment At 72 hour post-infection, extract 298M(2) still showed signs of significant viral inhibition, which was visible in the reduction on CPE Extracts 162M(4), 185M(4) and 397(1) showed signs of viral inhibition against HSV-1 within 24 hours post-infection, however these effects were not present at 72 hours post-infection Extract 495M(1) showed inhibition against both HSV-1 and VSV within 24 hours post-infection This effect was not present at the final experimental time-points and any inhibition found was negligible relative to the con-trols All other tested extracts were found to possess negligible inhibitive properties against both HSV-1 and
Figure 1 Representation of viral attachment/entry inhibition by marine extracts Viruses (VSV) were pre-incubated with test extract (100 μg/ml) Plates (Vero cells) were infected for one hour, after which plates were allowed to incubate for 24-36 hrs, until adequate plaques were observed Plates were stained with crystal violet staining and pictures were taken Plaques were counted and inhibition was determined relative
to controls Row 1: Extract 397(1), showing marked plaque reduction ( ≥ 80%) relative to the controls; Row 2: Extract 312(2), showing marked plaque reduction ( ≥ 90%) relative to the controls; Row 3: 338M(1), showing no marked plaque reduction (< 20%) relative to the controls; Row 4: Control of 0.1% DMSO.
Trang 7VSV This observation was based on CPE tracking, as
well as the production of infectious viruses
Extracts 331M(2) and 397M(1) showed significantly
high inhibition of CCV replication throughout the
dura-tion of the experiment, as determined by both reduced
CPE and virus production Extracts 298M(2) and 397(1)
showed significantly high inhibition of CCV replication
in CCO up to 48 hr post-infection, which decreased
slightly by 84 hr post-infection All remaining extracts
tested against CCV in CCO cells were determined to present no significant inhibition (P > 0.05) Viral titers and CPE determined for the remaining extracts were comparable to the control For SHRV, only extracts 397 (1) and 397M(1) showed signs of inhibition under these experimental conditions At 48 hr post-infection, 100% virus-induced CPE appeared in the control cells, as well
as in cultures treated with all other extracts, The cul-tures treated with extracts 397(1) and 397M(1) showed
Figure 2 Representation of viral replication inhibition by marine extracts Cells (CCO) were seeded into TC-12.5 cm2flasks and then infected with virus (CCV) at an MOI of 0.1 Following a 1-hr incubation, media was completely removed and infected cultures were subsequently incubated for approximately 3 days with 2.5 ml/flask of media containing extracts (100 μg/ml) Pictures were taken to track the progression of viral-induced CPE As shown, pictures were taken at 72 and 84 hours post-infection Extracts 397(1) and 397M(1) show > 90% viral inhibition, under the parameters of the experiment, relative to the control Extract 162M(4) shows no inhibition relative to the control.
Trang 8markedly reduced CPE (25-40%) These results were
confirmed by testing culture supernatants for viral titer
Discussion
Viral infections are the cause of many human and
ani-mal diseases that have tremendous economic impacts
The limited availability of antiviral measures, along with
the appearance of new virus types and drug-resistance
viral strains, have led scientists to expand their search
for novel drug candidates, recently turning back to
nat-ure The marine environment represents an almost
inex-haustible resource for antiviral drug leads, as oceans
encompass majority of the earth and its highly varying
dynamic nature has produce a wide range of organisms
that possess unique structures and produce distinctive
secondary metabolites In this study, in vitro assays were
established and employed to screen 20 marine
microor-ganism extracts for antiviral activity against four viral
isolates that are readily available in this laboratory
To properly test these marine extracts for antiviral
activity, highly concentrated starting materials and
broad dose-response studies provide the greatest
amount of information However, high concentrations of
marine extracts may be toxic to cell cultures To address
this, a set of experimental tests was performed to
deter-mine the safe and effective dose of these test extracts
for individual cell culture systems The concentration of
100 μg/ml was chosen as the maximum test
concentra-tion because drug-like molecules are typically sought to
have the desired effect at concentration less than or
equal to 100 μg/ml [26] In most drug development
cases, drug candidates that require concentration higher
than 100μg/ml are often discarded due to tolerance and
cytotoxicity issues, as well as cost effectiveness Also,
because these are extracts and not purified compounds,
the active molecule, if any, may be at a very low
concen-tration within the extract and a concenconcen-tration of 100
μg/ml may allow for any molecule present to produce
an antiviral effect The fact that most extracts remained
nontoxic throughout the 3-day experiment was
promis-ing All future experiments would rely on plaque assays
that have an incubation time of up to 72 hrs This time
requirement falls well within the range that these
extracts were shown to be nontoxic, thus validating the
use of these extracts in future experiments that test for
antiviral activity
The extracts were first tested for their ability to block
viral attachment/entry into the cells Viruses were
pre-incubated with test extracts at their maximum safe
con-centration to allow any interactions to take place that
may cause the neutralization of virus infectivity, possibly
by binding to and blocking the virus itself from adhering
to cells, or by blocking the cellular receptors that are
utilized by the virus to enter the cells This reduction of
viral infectivity was determined by a reduced number of viral plaque formations relative to controls containing only virus (Figure 1) The initial evaluation of these marine-extract specimens demonstrated that some of these extracts have antiviral potential
Results from these tests showed that these extracts provided a significantly higher amount of inhibition of VSV plaque formation than HSV-1 plaque formation, in Vero cells This phenomenon may be attributed to the nature of the envelope proteins of rhabdoviruses When comparing the inhibitive natures of these extracts, it was found that the extracts appear to show no consistent pattern of inhibition (Table 2) For HSV-1, the mamma-lian herpes virus, many of the extracts were not strongly preventative of viral entry or infectivity On the other hand, for the marine herpes virus CCV, many extracts showed inhibitive properties and a few were extremely potent Ecological pressures, such as competition for space, predation, symbiosis and tide variations, through-out thousands of years, originated the biosynthesis of complex secondary metabolites marine microorganisms, which in turn, allowed their adaptation to a competitive and hostile environment [3] This could lead to specula-tion that any viral inhibitive properties possessed by these marine microorganism extracts would be more suited against marine viruses Unfortunately, this propo-sal is negated by good-to-excellent anti-viral properties
of these marine microorganism extracts against the mammalian rhabdovirus VSV Many of the tested extracts demonstrated excellent VSV inhibition, but very few (in fact, only one) extracts were effective against the marine rhabdovirus SHRV A more likely explanation is that the results obtained herein are due to the specific nature of the antiviral mechanisms, producing differen-tial toxicity to individual viruses
Host cell composition and the factors present in each individual cell culture system may play a role in the effectiveness of each extract’s inhibition The cellular receptors available for viral attachment and entry may differ greatly between each cell type One may contain a virus-specific receptor that the components contained in
an extract can possibly bind to and block, while another cell culture system may possess this same receptor along with additional receptors with redundant func-tionality that might result in no apparent viral inhibi-tion Another contributing factor may be each cell line’s differential porosity to each extract’s components One extract’s antiviral element may be able to get into a spe-cific cell line easier than another, thus possibly produ-cing some replication inhibition in one cell line and not the other Further testing is needed to identify any of these contributing or limiting factors Future tests can
be specifically designed for a specific virus and host organism, thus eliminating any of these concerns
Trang 9In addition to viral attachment/entry, marine extracts
potentially possess other means of virus inhibition, such
as affecting viral replication after the cell has been
infected It was observed that some extracts showed
varying degrees of viral inhibition for HSV-1 during
early replication; however this did not last in later stages
of infection It is unknown at this time whether or not
the early inhibitory effects are transient due to the active
molecule being metabolized or degraded in culture, or if
the viral load increased to such an extent that the active
molecule was rendered ineffective For CCV, extracts
331M(2) and 397M(1) showed significantly high
inhibi-tion of CCV replicainhibi-tion throughout the durainhibi-tion of the
experiment This closely resembled the results from the
attachment/entry inhibition assays This significant
inhi-bition was seen in the CPE tracking, as well as the
pla-ques assay results These results may be reflecting the
extracts ability to prevent re-infection of the cells by
blocking the virus released into the media, however this
is unknown at this time It appears that extract 298M(2)
shows promise as a potential inhibitor of herpes virus
replication, as it show inhibitive properties to HSV-1
and CCV
Due to the small-scale of this initial study, there did
not appear to be strong correlations between the
amount of viral inhibition and the extract’s organism of
origin, however some general inferences were gained
For instance, extracts 312(2) (Bacillariophyceae cf
Odontella sp.), 313(2) (Bacillariophyceae Amphora sp.)
and 328(2) (Bacillariophyceae cf Odontella sp.) all
showed highly inhibitive properties for both HSV-1 and
VSV viruses, so one may infer that the marine diatom
has some general antiviral properties that are common
across diatom subspecies This statement is tentative
and will require more examination to corroborate
Extract 258M(1) from Cyanobacter sp also showed very
high levels of inhibition for both HSV-1 and VSV By
this same reasoning, one might infer that cyanobacteria
hold some general antiviral properties Other extracts
(162M(4), 298M(2), and 367M(1)) come from as-yet
unidentified bacterial origins, although they too showed
high levels of general antiviral activity for both HSV-1
and VSV It will be interesting to see if these extracts
also come from Bacillariophyceae, Cyanobacter or
another genus or species
There was also no detectable correlation between
sig-nificant viral inhibition due to active factor(s) that are
secreted (media extracts) or cell-based (whole organism
extracts) An equivalent number of cell-and
superna-tant-derived extracts were tested for their inhibitory
effects Both media and cell extracts alike showed
vary-ing levels of inhibition Equal numbers of cell-derived
and supernatant-derived extracts were shown to
pro-duce high to moderate levels of viral inhibition,
therefore these data do not elucidate whether or not the precise molecules within the extract that possess the antiviral properties Further studies, using direct com-parison of the media extracts from cultured marine microorganisms alongside whole-cell extracts of each organism, will be important for determining the location and differential production of soluble secreted or intra-cellular antiviral factors
There were likewise no correlations between the inhi-bition of viral plaque formation and cytotoxic activity There were several examples of compounds that were found to be cytotoxic and also inhibited virus plaque formation (298M(2), 313(2), 331M(3) and 438M(1)) These compounds would be less attractive targets for further development as antivirals unless they can be modified to reduce their non-specific cytotoxicity A contributing factor to underlying cytotoxicity may be the physical state of the starting extract Most extracts were liquids that ranged in color from a light-yellow to
a dark yellow, and even to light brown, with no particu-late matter However, there were some extracts, namely 313(2), 328(2), 460(1) and 491(1), that had distinct phy-sical properties These extracts were all cell-pellet extracted and their consistencies were more viscous and gelatinous than the other extracts, although they too did not contain particulate matter One notable exception was 313(2), a dark brown and gelatinous extract con-taining a substantial amount of particulate matter Extracts of this nature may somehow interfere with cel-lular stability or simply creates a hostile environment for cellular growth, producing toxicity In parallel, the same may be true about its antiviral effects Perhaps vis-cous extracts interact directly with the virus or cells, by simply creating a physical barrier that prevents viral attachment Further testing is needed to elucidate any answers
Taken together, the observed inhibition does not seem sufficient to suggest the application of these extracts as treatments of established viral infection Instead, these extracts may have potential use in prophylaxis to pre-vent infection, as well as prepre-venting the spread of infec-tion, due to the high level of inhibition displayed in the attachment/entry inhibition assays This is particularly pertinent in confined marine habitats that can be seeded with the active elements of these extracts in hopes of preventing the spread of viral diseases and decreasing mortality
Future studies can be focused on the isolation of the active elements contained in these extracts If the indivi-dual chemical components of the extracts can be identi-fied, then study of the exact chemical properties against specific viral genomic or proteomic components will be more convincing in demonstrating direct anti-viral mechanisms It is also possible that any of the observed
Trang 10antiviral effects resulted from synergy between
com-pounds found within the same extract Alternatively,
fractionation and isolation could have the opposite effect
of eliminating any antiviral potential This is because it
is well accepted that natural products are sometimes
efficacious due to additive or synergistic action between
multiple components within the matrix Therefore,
tak-ing a traditional Pharmaceutical Chemistry approach to
isolating individual chemicals may destroy the activity of
the complex mixture In any event, characterization of
the antiviral compounds and extracts, and elucidation of
their antiviral mechanisms and their parental marine
organisms, will be key in the discovery of new
com-pounds to be used as antiviral agents Isolation,
identifi-cation and characterization of marine compounds and
extracts from marine microorganisms with anti-viral
effects presents several potential implications, including
the important application as chemotherapeutic and/or
prophylactic agents of viral diseases of humans, lower
animals and marine animals, particularly in aquaculture
and conservation biology applications The
identifica-tion, chemical and genetic characterization of the active
principle(s) and moieties will facilitate the future
appli-cation of biotechnological procedures for increased
yields and cost-effective production
Conclusions
Hawai’i represents a geographical location where
biolo-gically useful products can be actively discovered [22]
New classes of organisms with novel characteristics are
constantly being discovered within the Hawaiian
archi-pelago Already, a few purified bioactive compounds and
over 2,000 unpurified crude extracts from a variety of
marine organisms, including sponges, bacteria and algae,
have been prepared Future studies will have access to
these previously established and readily available
resources The tests performed in this study have been
optimized and can be performed on a larger scale to
establish correlations and trends not seen in this
small-scale study The amount of viruses, host cell culture
sys-tems, as well as tested extracts can be greatly expanded
to yield more conclusive results With the knowledge
gained from large-scale tests, it may be possible to
opti-mize candidate search parameters of not only readily
available extracts, but also the search for new novel
organisms to be extracted, saving time and money Due
to the almost infinite amount of organisms that can be
examined and taking into consideration the
environ-mental pressures that cause similar organisms to evolve
and develop unique physical structures and secondary
metabolites, it is reasonable to conclude that discovering
novel antiviral drugs from marine microorganisms is
feasible and likely to be of considerable value for
emer-ging pharmaceutical needs
Acknowledgements The authors would like to thank the Thomas Hemscheidt laboratory for the assistance in preparation of marine microorganisms and extracts The author ’s would also like to thank Courtney Cox for technical assistance with cell cultures This research was supported in part by grants from the Centers for Oceans and Human Health (COHH) program, of the National Institutes of Environmental Health Sciences (P50ES012740), National Institutes of Health, and the National Science Foundation (OCE04-32479 and OCE09-11000) Author details
1 Department of Tropical Medicine, Medical Microbiology and Pharmacology, John A Burns School of Medicine, University of Hawaii at Manoa, 651 Ilalo Street, BSB Suite 320, Honolulu, HI, 96813, USA 2 Department of Public Health Sciences, John A Burns School of Medicine, University of Hawaii at Manoa, 1960 East West Road, BIOMED D104K, Honolulu, HI, 96822, USA.
3 Center for Marine Microbial Ecology and Diversity, 1680 East West Road, POST 105 University of Hawaii at Manoa, Honolulu, HI, 96822, USA Authors ’ contributions
JY carried out the cytotoxicity assays, the viral attachment/entry inhibition assays and the viral replication inhibition assays, as well as drafted the manuscript YY participated in the initial experimental tests and data analysis
of the study as well as provided useful technical input for assay protocols.
RB provided the extracts that were made previously for another study, as well as provided necessary information regarding the origin and preparation
of the extracts HTR provided marine isolates for the cultures and extracts YL was the principle investigator of this project and designed and conceived of the study, and participated in its coordination, and data analysis and manuscript revision All authors read and approved the final manuscript Competing interests
The authors declare that they have no competing interests.
Received: 1 July 2010 Accepted: 7 August 2010 Published: 7 August 2010
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